title: Ribosomal protein S5e is implicated in translation initiation through its interaction with the N-terminal domain of initiation factor eIF2α.
abstract: A key step of translation initiation in eukaryotes is formation of the 48S preinitiation complex (PIC) containing the 40S ribosome, a set of eukaryotic initiation factors (eIFs), mRNA, and initiator Met-tRNA interacting with mRNA start codon; however, the PIC structure remains substantially unknown. Here, we apply formaldehyde-induced protein-protein crosslinks to identify contacts between ribosomal protein S5e (rpS5e, "e" stands for "eukaryotic") and eIFs within the mammalian PIC, assembled on either model canonical or IRES-containing mRNA. Using immunoblotting and mass spectrometry, we show that with both types of mRNA, rpS5e crosslinks to eIF2α. Comparative analysis of peptides resulting from trypsinolysis of the crosslinked proteins before and after crosslink reversal reveals crosslinked peptides in the N-terminal parts of rpS5e and eIF2α. Application of these data to a model PIC structure obtained with the use of available structures indicates that eIF2α undergoes major conformation rearrangements to enable contacts of the factor with rpS5e. These contacts are suggested to maintain the correct positioning of eIF2α relative to other PIC components; this could be essential for start-codon selection by the PIC.
authors.affiliation: Laboratory of Ribosome Structure and Functions, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk, 630090 (Russia); Department of Molecular Biology, Novosibirsk State University, Ulitsa Pirogova 2, Novosibirsk, 630090 (Russia).
authors.forename: Dmitri
authors.initials: D
authors.lastname: Sharifulin
authors.forename: Elena
authors.initials: E
authors.lastname: Babaylova
authors.forename: Olga
authors.initials: O
authors.lastname: Kossinova
authors.forename: Yulia
authors.initials: Y
authors.lastname: Bartuli
authors.forename: Dmitri
authors.initials: D
authors.lastname: Graifer
authors.forename: Galina
authors.initials: G
authors.lastname: Karpova
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018387:Codon, Initiator; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D016174:Hepacivirus / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational / Q000502:physiology*; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*; D054682:Ribosome Subunits, Small, Eukaryotic / Q000737:chemistry / Q000378:metabolism
chemical_list: D018387:Codon, Initiator; D015852:Eukaryotic Initiation Factor-2; D012333:RNA, Messenger; D012269:Ribosomal Proteins; D006160:Guanosine Triphosphate
keywords: 40S ribosomes; initiation factor eIF2alpha; peptides; protein-protein interactions; ribosomal protein S5